CN105646656B - 灰星鲨软骨蛋白抗氧化肽及其用途 - Google Patents
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Abstract
本发明公开了一种灰星鲨软骨蛋白抗氧化肽及其用途,本发明以灰星鲨软骨为原料,通过总蛋白提取、丙酮分级沉淀、胰蛋白酶酶解得酶解液,酶解液采用超滤、离子交换树脂层析、凝胶柱层析和反相高效液相色谱分离纯化得到抗氧化肽Gly‑Glu‑Arg‑Glu‑Ala‑Asn‑Val‑Met,ESI‑MS测定分子量为905.00 Da;制备的高活性抗氧化肽具有较强的自由基清除活性和良好的脂质过氧化抑制作用,可以作为药品、保健食品或食品添加剂等进行开发。
Description
技术领域
本发涉及一种动物软骨活性肽,具体涉及一种灰星鲨软骨蛋白抗氧化肽及其用途。
背景技术
灰星鲨(Mustelus griseus)俗名沙条,又称灰貂鲨,为软骨鱼纲真鲨目皱唇鲨科星鲨属的一种。分布于西北太平洋区,包括中国南海、东海、黄海、台湾等海域,以及韩国釜山和木浦和日本南部等海区。
灰星鲨胆和肝、肉、皮、鱼鳍等均能入药。灰星鲨皮具有解鱼虾毒和消食化积等功效。灰星鲨肉具有健脾益气,祛瘀消肿,滋补强壮等功效,能够治疗体虚,伤口愈合缓慢,脾虚浮肿等疾病。灰星鲨翅具有滋补强壮,益气,补虚等功效。灰星鲨皮具有消积,解诸鱼毒等功效。灰星鲨胆具有清热解毒,消肿,活血等功效。灰星鲨骨具有祛风除湿,健脾止泻,活血止痛等功效。灰星鲨心具有健脾益胃,消痰化饮,益气开膈等功效。能够治疗因脾胃虚弱引起的内停痰饮,饮食不化,胸腕胀满,呕吐泄泻等症。
发明内容
本发明所要解决的技术问题是提供一种能够清除自由基和抑制脂质过氧化作用的灰星鲨软骨蛋白抗氧化肽。
本发明为解决上述技术问题所采取的技术方案为:一种灰星鲨软骨蛋白抗氧化肽:该灰星鲨软骨蛋白抗氧化肽,其特征在于该抗氧化肽为八肽化合物,氨基酸序列为Gly-Glu-Arg-Glu-Ala-Asn-Val-Met (GEREANVM),ESI-MS测定分子量为905.00 Da。
该灰星鲨软骨蛋白抗氧化肽的制备方法为:
1)灰星鲨软骨总蛋白粗提物的制备:称取灰星鲨软骨切成小碎块,加入适量蒸馏水,在高速组织捣碎机中匀浆至糊状,将糊状的灰星鲨软骨浸于4~6倍体积的1.0 mol/L盐酸胍溶液中(含0.02 mol/L MES和0.02 mol/L EDTA,pH 7~8),于4 ℃下搅拌抽提36~48h。提取液在4 ℃下以4000~6000 r/min离心15~25 min,弃残渣收集上清液;上清液继续在4 ℃、10000~12000 r/min离心15~25 min,取上清液用0.22 μm微孔滤膜过滤除去不溶性杂质。将滤液装入分子截留量为8 000的透析袋中,用Tris-HCl(pH 7.6)缓冲液于4 ℃下透析24~48 h,透析袋内溶液即为灰星鲨软骨总蛋白粗提物。
2)灰星鲨软骨丙酮分级沉淀蛋白的制备:取灰星鲨软骨总蛋白粗提物在冰浴下缓慢加入预冷丙酮至丙酮浓度为30%,在-20 ℃下静置4~6 h后,于4 ℃、10000~12000 r/min高速离心20 min,取上清液,加入预冷丙酮至溶液最终丙酮浓度为60%,获得灰星鲨软骨60%丙酮沉淀蛋白,冻干,即为60%丙酮分级沉淀蛋白。
3)灰星鲨软骨丙酮分级沉淀蛋白的酶解:取灰星鲨软骨60%丙酮分级沉淀蛋白按照料液比1g:3~5 mL溶于Tris-HCl缓冲液(0.05mol/L,pH 7.5~8.5),按照60%丙酮分级沉淀蛋白重量的2.0~3.0%加入胰蛋白酶(1.9×104 U/g),于40 ℃酶解3~5 h,然后于90 ℃、10 min灭酶活,酶解液于4 ℃、10000~12000 r/min离心15~25 min,弃残渣收集上清液,得软骨蛋白酶解液。
4)灰星鲨软骨蛋白抗氧化肽的制备:将上述酶解液采用3 kDa超滤膜进行超滤处理,收集分子量小于3 kDa部分,冻干,得超滤酶解物;将超滤酶解物次经离子交换树脂层析、凝胶柱层析和反相高效液相色谱(RP-HPLC)纯化,得抗氧化肽。
作为优选,所述的步骤1)中的灰星鲨为灰星鲨(Mustelus griseus)。
作为优选,所述步骤4)的离子交换树脂层析、凝胶柱层析和RP-HPLC纯化的具体过程为:
离子交换树脂层析:将上述超滤酶解物溶于双蒸水配成浓度为45~55 mg/mL的溶液,经过DEAE-52离子交换树脂层析柱,用水、0.1 mol/L、0.5 mol/L和1.0 mol/L NaCl溶液进行洗脱,流速为0.6~0.8 mL/min,洗出溶液每3 min收集一管并于280 nm检测,按峰合并试管内溶液,比较各峰的DPPH自由基和羟基自由基的清除活性,选择活性最强组分冻干,即为离子交换层析酶解物;
凝胶柱层析:将上述离子交换层析酶解物溶于双蒸水配成浓度为45~55 mg/mL的溶液,经过葡聚糖凝胶Sephadex G-15柱层析分离,用双蒸水进行洗脱,流速为0.6~0.8mL/min,洗出溶液每3 min收集一管并于280 nm检测,按峰合并试管内溶液,比较各峰的DPPH自由基和羟基自由基的清除活性,选择活性最强组分冻干,即为凝胶层析酶解物;
RP-HPLC纯化:将上述凝胶层析酶解物用双蒸水配成80~100 μg/mL的溶液,利用RP-HPLC进行纯化,根据对DPPH自由基和羟基自由基的清除活性得1个高活性抗氧化肽Gly-Glu-Arg-Glu-Ala-Asn-Val-Met (GEREANVM),ESI-MS测定分子量为905.00 Da。
再优选,所述RP-HPLC条件为:进样量10~15 μL;色谱柱Zorbax SB- C18(250 mm×4.6 mm,5 μm);流动相:水-乙腈梯度洗脱(0~32min乙腈浓度由0匀速升至50%);洗脱速度0.6~1.0 mL/min;紫外检测波长280 nm。
本发明还提供灰星鲨软骨蛋白抗氧化肽的用途:由于其具有安全无毒副作用、抗氧化活性强和易于消化吸收等优点,因而可以作为药品、保健食品和食品的添加剂。
与现有技术相比,本发明所提供的灰星鲨软骨蛋白抗氧化肽对DPPH自由基、羟基自由基、ABTS自由基和超氧阴离子自由基具有良好的清除作用;同时,Gly-Glu-Arg-Glu-Ala-Asn-Val-Met (GEREANVM)亦显示出良好的脂质过氧化抑制作用;Gly-Glu-Arg-Glu-Ala-Asn-Val-Met (GEREANVM)具有安全无毒副作用、抗氧化活性强和易于消化吸收等优点,可以作为药品、保健食品和食品的添加剂。
附图说明
图1是本发明的DEAE-52离子交换树脂层析图。
图2是本发明的葡聚糖凝胶Sephadex G-15层析图。
图3葡聚糖凝胶Sephadex G-15制备酶解物的RP-HPLC分析。
图4 Gly-Glu-Arg-Glu-Ala-Asn-Val-Met (GEREANVM)的质谱图。
图5 Gly-Glu-Arg-Glu-Ala-Asn-Val-Met (GEREANVM)的抗脂质氧化能力。
具体实施方式
以下结合附图实施例对本发明作进一步详细描述。
实施例:
一种灰星鲨软骨蛋白抗氧化肽的制备方法,制备工艺流程如下:双髻鲨软骨"软骨总蛋白"软骨60%丙酮沉淀蛋白"胰蛋白酶酶解"酶解物"超滤"离子交换层析"凝胶过滤层析"高效液相色谱制备"抗氧化肽。
1)灰星鲨软骨总蛋白粗提物的制备:称取灰星鲨(Mustelus griseus)软骨切成小碎块,加入适量蒸馏水,在高速组织捣碎机中匀浆至糊状,将糊状的灰星鲨软骨浸于4倍体积的1.0 mol/L盐酸胍溶液中(含0.02 mol/L MES和0.02 mol/L EDTA,pH 7.0),于4 ℃下搅拌抽提48 h。提取液在4 ℃下以5000 r/min离心20 min,弃残渣收集上清液;上清液继续在4 ℃、12000 r/min离心20 min,取上清液用0.22 μm微孔滤膜过滤除去不溶性杂质。将滤液装入分子截留量为8 000的透析袋中,用Tris-HCl(pH 7.6)缓冲液于4 ℃下透析48 h,透析袋内溶液即为灰星鲨软骨总蛋白粗提物。
2)灰星鲨软骨丙酮分级沉淀蛋白的制备:取灰星鲨软骨总蛋白粗提物,在冰浴下缓慢加入预冷丙酮至丙酮浓度为30%,在-20 ℃下静置5 h后,于4 ℃、10000 r/min高速离心20 min,取上清液,加入预冷丙酮至溶液最终丙酮浓度为60%,获得灰星鲨软骨60%丙酮沉淀蛋白,冻干,得60%丙酮分级沉淀蛋白。
3)灰星鲨软骨丙酮分级沉淀蛋白的酶解:取灰星鲨软骨60%丙酮分级沉淀蛋白按照料液比1g:4mL溶于Tris-盐酸缓冲液(0.05mol/L,pH 8.0),按照60%丙酮分级沉淀蛋白重量的2.5%加入胰蛋白酶(1.9×104 U/g),于40 ℃酶解4 h,然后于90 ℃、10 min灭酶活,酶解液于4 ℃、12000 r/min离心20 min,弃残渣收集上清液,得软骨蛋白酶解液。
4)灰星鲨软骨蛋白抗氧化肽的制备:将上述酶解液采用3 kDa超滤膜进行超滤处理,收集分子量小于3 kDa部分(MP60-I),得到超滤酶解液,将超滤酶解液依次经离子交换树脂层析、凝胶柱层析和反相高效液相色谱(RP-HPLC)纯化,得抗氧化肽。
①离子交换树脂层析:将MP60-I溶于双蒸水配成浓度为50 mg/mL的溶液,经过DEAE-52离子交换树脂层析柱,用水、0.1 mol/L、0.5 mol/L和1.0 mol/L NaCl溶液进行洗脱,流速为0.6 mL/min,洗出溶液每3 min收集一管并于280 nm检测,按峰合并试管内溶液,比较各峰的DPPH自由基和羟自由基清除能力,选择抗氧化能力最强组分冻干,即为离子交换层析酶解物MP60-3(图1);
②凝胶柱层析:将MP60-3溶于双蒸水配成浓度为50 mg/mL的溶液,经过葡聚糖凝胶Sephadex G-15柱层析分离,用双蒸水进行洗脱,流速为0.6~0.8 mL/min,洗出溶液每3min收集一管并于280 nm检测,按峰合并试管内溶液,比较各峰的DPPH自由基和羟自由基清除能力,选择抗氧化能力最强组分冻干,即为凝胶层析酶解物MP60-3-1(图2);
③RP-HPLC纯化:将MP60-3-1用双蒸水配成80~100 μg/mL的溶液,利用RP-HPLC进行纯化(RP-HPLC条件为:进样量15 μL;色谱柱Zorbax SB- C18(250 mm×4.6 mm,5 μm);流动相:水-乙腈梯度洗脱(0~32min乙腈浓度由0匀速升至50%);洗脱速度0.8 mL/min;紫外检测波长280 nm),根据对DPPH自由基和羟自由基的清除活性得1个高活性抗氧化肽DCPE-B(图3)。
④结构检测:收集DPPH自由基和羟自由基清除活性最高的抗氧化肽DCPE-B,经RP-HPLC检测为单一峰,利用蛋白/多肽序列分析仪测定氨基酸序列为Gly-Glu-Arg-Glu-Ala-Asn-Val-Met (GEREANVM),ESI-MS测定分子量为905.00 Da([M+H]+ 906.68 Da)(图4)。
将制得的灰星鲨软骨蛋白抗氧化肽Gly-Glu-Arg-Glu-Ala-Asn-Val-Met(GEREANVM)进行自由基清除实验和脂质过氧化抑制实验,实验结果表明:Gly-Glu-Arg-Glu-Ala-Asn-Val-Met (GEREANVM)对DPPH 自由基(EC50 2.76 mg/mL)、羟基自由基(EC500.22 mg/mL)、ABTS自由基(EC50 0.08 mg/mL)和超氧阴离子自由基(EC50 0.13 mg/mL);同时,Gly-Glu-Arg-Glu-Ala-Asn-Val-Met (GEREANVM)亦显示出良好的脂质过氧化抑制作用(图5)。
与现有技术相比,本发明所提供的灰星鲨软骨蛋白抗氧化肽对DPPH自由基、羟基自由基、ABTS自由基和超氧阴离子自由基具有良好的清除作用;同时,Gly-Glu-Arg-Glu-Ala-Asn-Val-Met (GEREANVM)亦显示出良好的脂质过氧化抑制作用;Gly-Glu-Arg-Glu-Ala-Asn-Val-Met (GEREANVM)具有安全无毒副作用、抗氧化活性强和易于消化吸收等优点,可以作为药品、保健食品和食品的添加剂。
最后,还需要注意的是,以上列举的仅是本发明的一个具体实施例。显然,本发明不限于以上实施例,还可以有许多变形。本领域的普通技术人员能从本发明公开的内容直接导出或联想到的所有变形,均应认为是本发明的保护范围。
SEQUENCE LISTING
<110> 浙江海洋学院
<120> 灰星鲨软骨蛋白抗氧化肽及其用途
<130> zjou-wb-201511-401
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 8
<212> PRT
<213> 人工合成
<400> 1
Gly Glu Arg Glu Ala Asn Val Met
1 5
Claims (2)
1.灰星鲨软骨蛋白抗氧化肽,其特征在于:该抗氧化肽为八肽化合物,氨基酸序列为Gly-Glu-Arg-Glu-Ala-Asn-Val-Met,ESI-MS测定分子量为905.00Da;
所述抗氧化肽对DPPH自由基的EC50为2.76mg/mL,对羟基自由基的EC50为0.22mg/mL,对ABTS自由基的EC50为0.08mg/mL,对超氧阴离子自由基的EC50为0.13mg/mL。
2.权利要求1所述的灰星鲨软骨蛋白抗氧化肽的用途,其特征在于:该抗氧化肽用于制备抗氧化保健食品或食品添加剂的用途。
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